Legionella pneumophila is an opportunistic pathogen of humans that can cause a severe, potentially fatal, pneumonia called Legionnaire's disease. Legionella infect human alveolar macrophages and survive intracellularly by using a type IV secretion apparatus to inject effector proteins into the host cell cytosol. These effector proteins allow L. pneumophila to evade transport to the lysosome and recruit host cell ER-derived vesicles to the Legionella-containing vacuole (LCV). A host cell SNARE protein, Sec22b, normally facilitates fusion of ER-derived vesicles with target membranes such as the Golgi. However, in Legionella infected cells, Sec22b localizes to the LCV within 30 min of infection. Host ER-derived vesicles bind and fuse to the LCV resulting in vacuole remodeling into a replication-competent organelle. The hypothesis of this proposal is that Sec22b plays a role in the recruitment and fusion of ER- derived vesicles to the LCV.
Specific aim 1 will use a yeast 2-hybrid approach and GST- pull down assays to identify bacterial proteins that bind to Sec22b.
Specific aim 2 will identify host cell proteins that play a role in vesicle binding and fusion.
Specific aim 3 will determine if the molecules identified in Specific Aims 1 and 2, or additional host cell molecules, facilitate the fusion of ER-derived vesicles to the LCV. Understanding the molecular mechanism of vesicle recruitment and fusion to the LCV is important for determining how L. pneumophila manipuates host cells to establish a replication-competent intracellular niche. In addition, these studies will likely enhance our understanding of the mechanisms governing vesicular transport in macrophages. Finally, completion of the specific aims by underrepresented students at CSUN will produce a new generation of research-oriented scientists trained for biomedically important graduate careers.
Legionnaire's disease is estimated to affect 25,000 people annually, leading to approximately 4,000 deaths per year. This project will identify molecules important for Legionella pneumophila's ability to survive and multiply in host cells. These molecules may be novel therapeutic targets for clinical intervention.
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